1. Field of the Invention
This invention generally relates to methods for altering surface characteristics of microspheres. Certain embodiments include coupling an enolic acid to the microsphere to modify the surface characteristics of the microsphere such that a reagent can be coupled to the microsphere via the enolic acid.
2. Description of the Related Art
The following descriptions and examples are not admitted to be prior art by virtue of their inclusion within this section.
Spectroscopic techniques are widely employed in the analysis of chemical and biological systems. Most often, these techniques involve measuring the absorption or emission of electromagnetic radiation by the material of interest. One such application is in the field of microarrays, which is a technology exploited by a large number of disciplines including the combinatorial chemistry and biological assay industries. One company, Luminex Corporation of Austin, Tex., has developed a system in which biological assays are performed on the surface of variously colored fluorescent microspheres. One example of such a system is illustrated in U.S. Pat. No. 5,981,180 to Chandler et al., which is incorporated by reference as if fully set forth herein. In such a fluid flow device, microspheres are interrogated by laser excitation and fluorescence detection of each individual microsphere as it passes at relatively high speed through a detection zone. Measurement data generated by such a system may be easily exported to a database for further analysis.
Assays based on fluorescent microspheres for multiplexed analysis have been also reported by several groups and individuals as described by Fulton et al., Clin. Chem., 1997, 43, 1749-1756; Kettman et al., Cytometry, 1998, 33, 234-243; McDade et al., Med. Dev. Diag. Indust., 1997, 19(4), 75-82; McHugh, Methods Cell Biol., 1994, 42, 575-595; and Nikiforov et al., Nucleic Acid Res., 1994, 22, 4167-4175; and U.S. Pat. No. 5,736,330 to Fulton, U.S. Pat. No. 6,046,807 to Chandler, U.S. Pat. No. 6,057,107 to Fulton, U.S. Pat. No. 6,139,800 to Chandler, U.S. Pat. No. 6,268,222 to Chandler et al., U.S. Pat. No. 6,366,354 to Chandler, U.S. Pat. No. 6,411,904 to Chandler, and U.S. Pat. No. 6,449,562 to Chandler et al., which are incorporated by reference as if fully set forth herein.
In the above-mentioned systems, fluorescent dyes are absorbed into the microspheres and/or bound to the surface of the microspheres. The dyes are chosen based on their ability to emit light in the wavelength of a chosen detection window of the system. Further, the detection windows are spaced apart by a number of wavelengths, and the dyes are designed to minimize the overlap of a dye's fluorescent signal within adjacent detection windows. By employing two detection windows and two dyes, each at 10 different concentrations, there would thus be 100 fluorescently distinguishable microsphere sets.
In the last three decades, advancements in the fields of affinity chromatography, solid-phase synthesis, and immobilization of bio-macromolecules, such as proteins, oligonucleotides and the like, have led to microsphere-based biomedical applications. For example, one or more biomolecules may be bound to the surface of microspheres. The one or more biomolecules are selected based on the specific assay to be carried out. For example, one population of microspheres may include different subsets of microspheres, each coupled to a different antigen. The subsets may be combined with a sample, and the assay may be performed to determine which antibodies are present in the sample. The biomolecule(s) that are bound to the microspheres may include any biomolecules known in the art.
The immobilization of biomolecules or any other such entities can be achieved by coupling by (a) ionic interactions; (b) adsorption; (c) complexation (e.g. “metal-coordination” mediated coupling); and (d) covalent bond formation between active/stable reactive groups on the surface and specific functional groups on the entity to be immobilized. For example, particles (e.g., micro- and nano-spheres; nanotubes; metal particles including one or more metals with any size, shape, or composition; semiconductor particles; molecularly imprinted polymers (MIPS); magnetic particles; and other dyed materials) and microtiter plates are common solid matrices in many immobilization systems. Preparing and maintaining the active, functionalized surface of the solids are important to assure immobilization of biological material for development of a sufficiently sensitive assay. Current procedures for immobilization of biomolecules on solid surfaces generally involve reactions of activated carboxyl, amino-, hydroxyl- or thiol-groups on the solid surfaces with the biomolecules. After activation of, or introduction of a functionalized spacer to, these groups, the activated groups provide sites on the solid surface for direct attachment of the biomolecules.
Currently used groups for providing direct attachment sites, however, have a number of disadvantages. For example, most of these functional groups (such as N-hydroxysuccinimide (NHS) esters, isothiocyanates, etc.) are prone to hydrolysis in an aqueous environment and become non-reactive (i.e., chemically inactive) in a matter of less than an hour. Therefore, such functional groups may undesirably exhibit time-dependent variations in the quantity, repeatability, and uniformity with which biomolecules may be attached to the surface of solids using these functional groups.
Reactive or functionalized microspheres are conventionally produced via copolymerization of suitably functionalized monomers or via chemical modification of preformed microspheres. Post-functionalization is a popular method for preparing reactive particles as earlier described by Upson, (J. Polym. Sci., Polym. Symp., 1985, 72, 45, which is incorporated by reference as if fully set forth herein.
More recent work on the production and evaluation of a variety of tailor-made particles has been reported by several groups including Margel, et al., (J. Polym. Sci., 1991, A-29, 347-355; Anal. Biochem., 1981, 128, 342-350), Ugelstad et al., (Makromol. Chem., 1979, 180, 737-744; Adv. Colloid Interface Sci., 1980, 13, 102-140), and Rembaum et al. (Br. Polym. J., 1978, 10, 275-280; J. Macromol. Sci. Chem., 1979, A-13, 603-632), which are incorporated by reference as if fully set forth herein. A review by R. Arshady, Biomaterials, 1993, 14, 5-15, which is also incorporated by reference as if fully set forth herein, describes the synthesis and physico-chemical properties of reactive and labeled microspheres.
Fray et al., Bioconjugate Chem., 1999, 10, 562-571, which is incorporated by reference as if fully set forth herein, have reported a strategy in which particles are pre-activated with hydrolysis-resistant aldehyde functional groups, but low reaction yields of less than 8% have been observed with these microspheres. U.S. Pat. No. 6,146,833 to Milton, which is incorporated by reference as if fully set forth herein, describes a reaction between an acyl fluoride activated polymer-surface and an amino derivatized biomolecule at room temperature. The use of fluorophenyl resins in the solid phase synthesis of amides, peptides, hydroxamic acids, amines, urethanes, carbonates, sulfonamides, and alpha-substituted carbonyl compounds has been described in International Publication No. WO 99/67228 to Clerc et al., which is incorporated by reference as if fully set forth herein.
Medvedkin et al., Bioorg. Khirn., 1995, 21(9), 684-690, which is incorporated by reference as if fully set forth herein, illustrates using sulfo-tetrafluorophenyl activated esters in peptide synthesis and demonstrates their reactivity combined with good stability under aqueous storage conditions. Apparently, the pre-activation of a polystyrene surface with this reagent has not yet been reported.
Hoechst, in German Patent No. DE 960,534 to Heyna et al., which is incorporated by reference as if fully set forth herein, claimed the use of reactive vinyl sulfone (VS)-modified dyes for dyeing of cellulose and wool fibers in 1950. A review by Siegel provides a complete account of reactive dyes based on VS and its protected 2-sulfatoethyl and 2-thiosulfatoethyl sulfones (E. Siegel in The Chemistry of Synthetic Dyes, Vol. VI, (Ed. K Venkataraman); 2-108, Academic Press, 1972, which is incorporated by reference as if fully set forth herein). U.S. Pat. No. 5,414,135 to Snow et al., which is incorporated by reference as if fully set forth herein, describes modification of proteins with PEG-supported VS.
The most frequently used method to immobilize biomolecules (such as oligonucleotides, proteins, and carbohydrates) onto fluorescent microspheres is by activating carboxy groups present on the surface of the microspheres. The activation requires excess N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC) and a coupling pH of 4 to 6. The reaction between the carbodiimide and carboxyl functional groups forms an activated O-acylurea derivative reaction intermediate. A subsequent nucleophilic attack of the reaction intermediate by the primary nitrogen of the amino-groups of the biomolecule being attached to the microspheres releases the substituted urea and produces an amide linkage between the reaction intermediate and the biomolecule.
There are, however, a number of disadvantages to such activation of the carboxy groups. For example, the reaction intermediate has an extremely short half-life and rapidly undergoes hydrolysis or rearranges to produce the N-acylurea adduct. In addition, the optimum pH for the formation of O-acylurea is about 4-5. However, the primary amino group of the nucleophile is predominantly protonated at a pH of about 4-5 and is thus mostly unreactive. These limitations of the reaction intermediate can severely restrict coupling yields of biomolecules to microspheres. Furthermore, at low pH, nucleic acid bases of a biomolecule may undergo intensive protonation. Such protonation induces DNA melting that exposes the hydrophobic core of the helix thereby facilitating nonspecific hydrophobic interactions of the helix with the solid matrix of the micro spheres.
Despite these drawbacks, EDC-mediated coupling is currently the major mode of covalent immobilization of biomolecules to solid surfaces as described by Hermanson, G. T., in Bioconjugate Techniques, Academic Press, NY, 1996; Frey, A. et al., Bioconjugate Chem., 1999, 10, 562-571; Gilles, M. A. et al., Anal. Biochem., 1990, 184, 244-248; Chan V. W. F. et al., Biochem. Biophys. Res. Communications, 1988, 151(2), 709-716; and Valuev, I. L. et al., Biomaterials, 1998, 19, 41-43, which are all incorporated by reference as if fully set forth herein.
For combinatorial libraries, building blocks such as malonic acids, dihydroxy benzoic acid, hydroxy phenyl acetic acid, pyroline carboxylic acids, bromodihydroxy benzoic acids, 3-oxo-1-indancarboxylic acid, 3-nitrophenyl acetic acid, and 3,4-difluoro benzoic acid have been reported by, for example, Lin, R. et al., in J. Am. Chem. Soc., 2002, 124, 7678-7680, which is incorporated by reference as if fully set forth herein.
Some molecules that can be incorporated into polymers to modify the surface characteristics of the polymers have been reported and are shown below.
Organic reactions using polymer supported catalysts, reagents or substrates are known as described by, for example, Hodge, P. in “Synthesis and separations using functional polymers,” Editors, Sherrington, D. C. & Hodge, P., 1988, John Wiley, 44-113, which is incorporated by reference as if fully set forth herein.
Polymer supported phenolic compounds are known. For example, polymer supported tetrafluoro phenol is now used as an activated resin for chemical library synthesis as described by Salvino, J. M. et al., J. Comb. Chem., 2000, 2, 691-699, which is incorporated by reference as if fully set forth herein.
Boronic acid is routinely incorporated into synthetic receptors for the complexation of saccharides and other guests that possess 1,2 and 1,3 diol functionality, as described by Czarnik, A. W. et al., J. Am. Chem. Soc. 1992, 114, 5874, Shinkai, S. J., J. Chem. Soc. Chem. Commun., 1994, 477, and Geert-Jan Boons et al., Tetrahedron Lett., 200, 41, 6965, which are incorporated by reference as if fully set forth herein. Boronic acids have also been incorporated into a chemical affinity system for the purification of proteins, as described by Bergseid, M. et al., in Biotechniques, 2000, 29, 1126, which is incorporated by reference as if fully set forth herein. The use of various boronic acids to link two entities together has been disclosed in U.S. Pat. No. 6,008,406 to Stolowitz, U.S. Pat. No. 6,075,126 to Stolowitz et al., U.S. Pat. No. 6,124,471 to Stolowitz et al., U.S. Pat. No. 6,462,179 to Stolowitz et al., and U.S. Pat. No. 6,630,577 to Stolowitz et al., which are incorporated by reference as if fully set forth herein.
Acidic functional groups have also been added to glass surfaces as described by, for example, Geiger, F. M. et al., J. Am. Chem. Soc., 2004, 126, 11754, which is incorporated by reference as if fully set forth herein.
Accordingly, it would be advantageous to develop a method for altering the surface characteristics of a microsphere without one or more of the disadvantages described above such as time-dependent variations in the attachment of biomolecules to the surface of microspheres due to hydrolysis of the functional groups used to attach the biomolecules.